兽疫链球菌透明质酸分解酶基因的敲除

以透明质酸分解酶基因（Hyl）为改造目标, 利用同源重组技术获得 Hyl基因敲除的重组菌。首先以兽疫链球菌的基因组DNA为模板扩增出部分透明质酸分解酶基因（Hyl-1），然后将其克隆到载体pMD19-T上，再以质粒pUC19为模板, PCR扩增得到氨苄青霉素抗性基因,通过反向PCR将其插入Hyl-1基因的中部，得到基因敲除载体pMD19T-SA。该载体与质粒pBR322分别用EcoRⅠ、PstⅠ双酶切后进行连接,得到基因敲除的重组载体pBR322 SA，PCR 及限制性酶切分析,构建的敲除载体与设计结果相符。最后,利用这两种敲除载体通过同源重组技术得到一株重组菌,经PCR 及酶活鉴定,这株菌的Hyl 基因已缺失。

Gene Knocking Out of Hyaluronidase in Streptococcus zoopidemics

To construct knockout vectors containing ampicillin resistant gene and partial sequence of hyaluronidase gene(Hyl) so that Hyl can be knock out by transforming the plasmid into Streptococcus zoopidemics mutans. First, partial sequence of Hyl ( Hyl-1 ) was cloned into the vector of pMD19-T by using DNA of Streptococcus zoopidemics as template,and then a knockout vector pMD19T-SA was constructed, in which Hyl-1 gene was disrupted by inserting ampicillin resistant gene( Amp)from reverse PCR. As expected, the vector was proved to be consisted of Hyl-1-Amp-Hyl-1-pMD19-T. Thereafter, DNA fragment of Hyl-1-Amp-Hyl-1 was subcloned into pBR322 vector,the resulting construct was then checked by PCR and restriction analysis for the proper configuration of the knockout vector pBR322-SA. Both of the knockout vectors were used to transform Streptococcus zoopidemics and one recombinant was obtained in result. From results of PCR and Hyl activity assay, it was indicated that in the recombinant the Hyl gene was disrupted completely.